Selective and Differential Media

• Selective media inhibits the growth of certain species in a mixed sample, allowing the species of interest to grow.
• MacConkey agar is selective for Gram-negative rods.
• Crystal violet inhibits Gram-positive bacteria, and bile salts inhibit most non-intestinal bacteria in MacConkey agar.

Differential Media

• Differential media contain substances that microbes change in an identifiable way.

• Blood agar plates are an example of differential media as they tests for secreted toxins that attack red blood cells.

• Beta hemolysis produces a clear zone on blood agar plates.

• Alpha hemolysis produces a zone of greenish partial clearing on blood agar plates.

• Selective or differential media are used to detect or isolate a species from a mixed population.

Membrane Filtration

• Membrane filtration allows for the detection of low numbers of bacteria.
• A known volume is passed through a sterile filter,
• Microbes are captured and concentrated by filtration.
• Water or air can be filtered
• The filter is incubated on an appropriate agar medium.

Providing Appropriate Atmospheric Conditions

• Petri dishes in air are Aerobic, at least for surface colonies.
• Most obligate aerobes and facultative anaerobes can be incubated in air which contains approximately 20% O2.
• Broth cultures need to be shaken or "bubbled" to provide maximum aeration.
• Bacteria such as Neisseria and Haemophilus grow best with increased CO2 and/or low oxygen levels.
• Capnophiles require increased CO2.
• One method to incubate capnophiles is in a candle jar
• Microaerophilic cultures should be grown in agar stabs or stationary liquid medium.
• Anaerobes require lower O₂ concentrations than achieved by a candle jar.
• Anaerobes can be incubated in gastight container with chemical packet, or flushed with nitrogen or other gas, or work in a glovebox.

Methods to Detect and Measure Microbial Growth

• Direct cell counts measure total numbers, living plus dead.

• Direct microscopic count involves counting cells directly using a microscope.

• Cell-counting instruments like Coulter counters and flow cytometers can be used.

• The class includes general principles, approaches, specific treatments, and chemical treatments.

• In 1700, Leewenhoek demonstrated that microorganisms exist.

• In 1861, Pasteur disproved spontaneous generation and he and Koch independently showed MOs could cause disease.

• In 1865, Lister first used chemical treatment to block wound infection.

• Until the late 1800s, hospitals had high mortality rates from infections after surgeries.

• Childbirth was safer with a midwife.

• Physicians would not initially accept that they could pass diseases from one patient to the next.

• Understanding airborne microbes could infect open wounds took time.

Modern Hospitals

• Modern hospitals face tough challenges, combining high numbers of sick patients, a very high incidence of pathogens and with more patients with compromised immune responses

• Control of “Nosocomial” infections, now called Healthcare-Associated Infections, HAIs, is essential.

• Medical personnel need protection from diseases such as COVID and Ebola.

• Standard Precautions protocols and Transmission-Based Precautions protocols are in place.

• Special challenges exist in operating rooms and surgery.

• Food preparation and preservation need special protocols to control microorganisms.

• Control of preservation is more critical than ever due to mass production, broad distribution, and long-distance transport.

Key Definitions

• Sterilization involves removing or destroying all microorganisms and viruses
• A sterile item is free of microbes, including endospores, but not prions
• Disinfection involves eliminating most or all pathogens, but some viable microbes may remain.
• Disinfectants are chemicals used on inanimate objects known as germicides.
• Bactericides kill bacteria.
• Fungicides kill fungi.
• Virucides inactivate viruses.
• Antiseptics are chemicals used on living tissues.
• Decontamination reduces the number of pathogens to a safe level
• Includes washing, use of heat, or chemicals
• Sanitization substantially reduces the microbial population to meet accepted health standards that minimize the spread of disease.
• Preservation is the process of delaying spoilage of perishable products.

Food Preservation Techniques

• In choosing conditions of storage to slow growth, add bacteriostatic preservatives.
• Pasteurization uses brief heating to reduce the number of spoilage organisms and destroy pathogens without significantly changing the product.
• Cold pasteurization achieves the same result using pressure instead of heat.
• BSL-1. Procedures are for work with microbes not known to cause disease in healthy people
• BSL-2. Procedures are for work with moderate-risk microbes with limited transmission potential, like the biology research laboratories in UTEP
• BSL-3. Procedures are for work with pathogens that cause serious or potentially fatal disease through inhalation, special containment facilities at UTEP
• BSL-4. Procedures are for work with easily transmitted deadly pathogens

Daily Life Protocols

• Protocols in daily life include washing and scrubbing with soaps and detergents on hands, utensils, and countertops.
• It is worth noting that most commensals are deep in follicles and survive daily washing.
• Cooking foods to proper temperatures and storing perishables by refrigeration or freezing are are other routine precaution.
• Special care should be taken with known pathogens in poultry and salmonella.

Food preparation and preservation

• Food preparation and preservation concerns include storage of food for extended times and preventing contamination transmission of pathogens or toxins.
• Microbes protect against microbes through fermentation.
• Dominate ecosystem with “good” (edible, non-pathogenic) microbes
• Yoghurt and Cheese preserve milk.
• Also utilizing wine, sauerkraut, kombucha, and kimchi

Food Preparation Practices

• Food-processing facilities must maintain clean surfaces and machinery, relatively free of microorganisms.
• Must start with healthy animals and plants and comply with FDA regulations in U.S.

Food Preservation

• Food preservation by temperature includes refrigeration, freezing, cooking, and canning.
• Refrigeration and Freezing lengthen storage time but does not eliminate pathogens.
• Cooking and Canning involve killing organisms then sealing in a germ-proof container.
• High heat is needed for endospore destruction, which changes food characteristics.
• Some foods are combined with acid for “hot pack" pickling.
• If they are not properly treated, this creates for a perfect incubator for anaerobes, resulting in bulging cans or tops.

Pasteurization Methods

• Pasteurization uses an intermediate temperature that kills “'9most harmful bacteria" with less change to food, usually in conjunction with refrigeration.
• Cold pasteurization is a high-pressure based based method.
• Dehydration involves air drying using sun or low heat, vacuum drying (freeze), or by adding salting or sugaring.
• An addition of salt or sugar increases environmental solutes, drawing water out of cells and dehydrating.
• Drying stops microbial growth but does not reliably kill and cases of salmonellosis have come from dried eggs

Chemical Preservatives

• Chemical preservatives can prevent or retard spoilage by preventing oxidation of fatty acids

• Weak organic acids benzoic, sorbic, and propionic acids, affect cell membrane function and control molds and bacteria in foods.

• Nitrate and nitrite inhibit endospore germination and vegetative cell growth in processed meats and stop growth of Clostridium botulinum.

• Higher concentrations give meats pink color, but can be carcinogenic when converted to nitrosamines by cooking or intestinal bacteria.

• Filtration uses extremely small pores that won't let bacteria pass and is used for liquids and air.

• Irradiation can be extremely effective, can allow milk to be stored for a year at room temperature and keep fresh fruit from spoiling.

• There are cultural issues with irradiation due to nuclear and radiation concerns and unknown changes to highly irradiated proteins.

• Keeping food alive as long as possible involves utilizing natural immune defenses.

Water Treatment

• Filtration can be small batch, like backpacking.
• Sedimentation removes particles and parasite ova.
• Regulations in water facilities also require facilities to minimize DBPs and C. hominis after the addition of chlorine Chlorine reacts with naturally occurring chemicals, and potentially create disinfection by-products(DBPs) that that are linked to long-term health risks.
  • Some organisms are resistant to chemical disinfectants
• Cryptosporidium hominis is an water intestinal pathogen that causes diarrhea

Other Industries

• The other industries that are impacted are pharmaceuticals cosmetics, which deodorants must not carry microbial contamination, and hand cream and cleaners
• Selection of an effective antimicrobial procedure is complicated.
• An ideal method does not exist, and each has drawbacks.
• The choice of disinfection chemicals depends on the type and number of microbes, environmental conditions, risk of infection and composition of the item to be treated.

Resistant Microbes

• Multiple highly resistant microbes are bacterial endospores which are most resistant and only extreme heat or chemical treatment destroys them.
• Protozoan cysts and oocysts are resistant to disinfectants, excreted in feces, cause diarrheal disease if ingested and boiling can destroy them.
• Mycobacterium species have waxy cell walls, makes resistant to many chemical treatments, aid survival in air.
• Pseudomonas species: resistant to and can actually grow in some disinfectants and plain water.
• Non-enveloped viruses lack lipid envelope, are more resistant to disinfectants and alcohol hand sanitizers have no impact on them.

Microbes in the Population

• Only a fraction of the population dies during given time interval
• Takes more time to destroy a large population than a small one
• Removing some organisms by washing reduces time needed to sterilize or disinfect a product
• Scrubbing helps remove biofilms, important because organisms in a biofilm are resistant to disinfectants
• Decimal reduction time ,D value is equal to time required to kill 90% of population under specific conditions.
• Constant rate of killing requires the need to exceed the initial population of MOs to ensure sterization

Environmental Conditions

• Dirt, grease, and body fluids can interfere with heat penetration and action of chemicals, so thoroughly clean items

• Temperature and pH can influence effectiveness.

• Sodium hypochlorite bleach solution can kill suspension M. tuberculosis at 55°C in half the time as at 50°C and is even more effective at low pH

• If contact with body tissues under the skin occurs then instruments must be sterile like needles and scalpels. -If contact occurs with mucus membranes, but do not penetrate into internal body tissues, the item must be clean and free of microorganisms and viruses. -Okay in some endospores may remain, usually blocked by mucous membranes such as endoscopes and endotracheal tubes.

• For instruments and surfaces contact unbroken skin only a low risk if transimission and clean is preferred. -Examples are countertops stethoscopes, and blood pressure cuffs Some items require special consideration of sterilization and disinfection methods because heat can damage plastics and other materials Irridation provides alternative, but damages some types of plastic. Ethylene Dioxide gas and Moist heat are also alternatves to heat Moist heat and liquid chemical disinfectants cannot be used to treat moisture-sensitive material

• Reliability, safety, speed, cost, and non-toxicity are all considerations when deciding to use heat to sterilize or disinfect. Alternatives include filtration, irradiation, or chemical or high-pressure treatments on materials that cannot withstand heat treatment

Moist Heat

• Boiling can irreversibly denature proteins

• Boiling destroys most microorganisms and viruses, but does not sterilize, as endospores can survive

• Pasteurization destroys heat-sensitive pathogens and spoilage organism.

  • High-temperature-short-time (HTST) method uses 72°C milk for 15 seconds and 82°C for 20 secondsfor ice cream. -Ultra-high-temperature (UHT) method produces shelf-stable boxed juice and milk, known as "ultra-pasteurization", 140°C milk for a few seconds, then rapidly cooled.

• Autoclaving sterilizes using pressurized steam via increased pressure that raises steam temperature and kills endospores. -Sterilization is typically at 121°C, 15 psi for 15 minutes and for larger volumes takes longer. -Flash sterilization at higher temperature but shorter can be used -Destroying prions involves immersion in 1 M NaOH during or before autoclaving.

• The Commercial Canning Process uses industrial-sized autoclave called retort. -Procedure designed to destroy Clostridium botulinum endospores and reduce 1,012 endospores to only 1 (an 12D process). Critical becuase Virtually impossible to have that many endospores

• Endospores could germinate in canned foods is critcial becuase cells grow in low-acid anaerobic conditions and produce botulinum toxin(bulging lids)

• Commercially canned food is sterile. Endospores of some thermophiles may survive,but Usually not a concern because only grow at temperatures well above normal storage is not a concern Incineration oxidizes cell components using dry heat, such as flame.

• Incineration are used in microbiology laboratories, as are the wire loops continually reused to transfer bacterial cultures sterilized by flaming or heating in a benchtop incinerator.

• Incineration is also used to destroy medical wastes and contaminated animal carcasses

• Hot air ovens kill microbes are used by destroying ceil components and denaturing protrins Requires higher temperature and longer time than moist heat because dry heat takes longer to penetrate and is less efficient at killing microbes.

• Membrane filtration of a liquid uses pore sizes of .2 um to retain bacteria while allowing fluid and dissolved components to pass through

• Used extensively to remove organisms from heat sensitive fluids eg serum

• Apply vaccum to speed filtration

• Filtration can used to minimize respiratory infections

• HEPA filters remove nealy all microbes over 0.3 um

• Used in specialized hospital rooms, biological safety cabernets, and those caring for specific infections such as COVID-19 and M. tuberculosis

• Ioninzing Radiaton is a recent form of microbial control

• Includes gamma rays, Xrays Directly interferes and impacts the cytoplasmic membranes, thus destorying DNA Also can react with O2 and create reactive oxygen species Commonly used on heat sensitive and packaged materials Has great effectiveness. Apporved for use on a variety of foods, but some consumers refuse irradiated products

• Ultraviolet Ratiation destroys the microbes directly

• Also damages DNA in the process Best when used in air, water, and on surfaces

• It has poor prenetation

• Can’t act well with films, coverings, solids, or turbid liquids

• glass and plastic both block rays

• Careful usage is needed for users, as it have effects on skin and eyes

• Microwaves They can be used to make products lethal, however doesn’t not directly effect microorganisms, but the heat generate can do harm

• Uneven heating causes cells to survive in cooler packets.

• High Pressure Processing (HPP) is also referred to as pascalization or cold pasteurization

• Decreases the number of microorganisms in foods like fruit juice and guacamole

• Employs pressure up to 120,000 pound per square inch to break donw

• It denautures portiens and impacts cell permeability, causing cell destruction, but food still keeps most color, flavor, and freshness

Chemicals

Germicidal chemicals disinfetcs, and can somtimes sterilize Reacts irreversibly with protens, cytoplasmis membranes viruses, and DNA Exact mechanisms are porrly understood, but they react differently than heat, less reliable, but still helps when heat-sensitive materials are on hand

• Some are sufficiently none toxic to be used

• Germicidal chemicals disinfect and, sometimes, sterilize -Sterilants destroy all microbes and may contain sporocides -Apporved for use on heat-sensitive critical instruments

• High-level disinfectants destroy viruses, vegetative cells -Apporved se on semi-critical instruments , but do not reliably kill endospores

  • Intermediate-level disinfectants destroy vegetative bactera, mycobacteria, fungi, most viruses Approved for use on desinfectable that isn’t crirtical instruments-Low-level disinfectants destroy fungi, vegetative bactera, but not mycobracteria, and is effective at managing enveloped viruses Doesn’t destroy endospores, viruses, and is also most effective on surfaces

• 60% to 80% aqueous solutions of ethyl or isopropyl alcohol (ALCHOHOL SOLUTIONS)

• For destroying bacteria and fungi

• It isn’t reliable and won’t work on sporces, or non-enveloped viruses When doing work on surfaces, please note it denatures protiens Protens have to be fully dissolved with water, for full effect Commonly used as antiseptic and disinfectant due to no sidee effects on users and ease of use

• There are limitations It evaporates, so the work time must stay constant with whatever product being used Avoid damage on materials, like rubber and plastics

• ALDEYHDES-

• Has most compounds, glutaraldehyde, that deactivate portiens, making it effective as a sterilbant

• Most effective on liquids and in heat When using, it does take an extemely long period of time

• Always follow up and ensure you are wearing proper saewty equipment

• Less toxic, so is used more commenly to clean sensitive materials

• There can be an increase of cancer with the use

• Is effective at killing a range of bactiera

• Commonly used on the outside, to prepare for vaccines Biquanides

• Use as effective in anti sepitcs

• Stays near the membrane and on the skin, so that cells and new growth are always monitored and impacted

• It kills some vegetative bactiera, fungi, and some enveloped viruses

• It may be used on prescription mouthewases and topical skin

Ethlene Oxide

• For most products it is gas sterilant and a product that deals will heat/moisture items

• Must be done by someone expereinced and safe

• Includes matresses, tools used for electrical devices, and things artificial hips It can be applied in a special chamber (like Autoclave)

• Be careful, as there can be some limitations

• It could explode, be toxic, cause cancer, Must make sure elimation is done thorugh heating Cholring (Haolgen oxidizing aernts) They are used with ports/cells and always on surfaces

• The goal is to manage all cells and clean, to reduce endospores and other viruses, and to make it caustic to other and users

• Always wear gloves

• A bleach can always be effective as 1:100 solution

• They can react with compunds in organic systems and create carcinogens Iodine

• kills Vegetative, and unreliable on all existing spores

• it is a tincture

• it acts as an iodopher that is a new carrier, and is known to irritate less.

• Pseudomonas can be survivied in stock solution Silver combines with sulfhydrl to create protiens Silver is not as effect to be medically used

• Some ointments are still used so that Neisseria’s do not spread and gonorrhoeae infections do not contianute (Use antibiotics for the best control )

• FDA notes to always see for preservation options Ozone Unstable forms of oxygen , decompse quickly, so has to be generated Effective at replacing chlorine and always must be desifecting for drinking pools

• A phenolic will create an envirment that effects the Phenolic cell

• The phenol is best effective at killing all bateria types, but isn’t for viruses or other bacterias Always be effective in environments and not the others.

• There can be some residue

• Triclosan is effective for personal care and medical

Quaterany

• Some can be effective and cleaning services to eliminate and make surfaces safe.

• The goal is to reduces the tension

• The goal is to attack, create a charge, and then desory any surounding virsues

• A pseduomonas can be survivied Use preservatives -Chemically created can prevent growth and shelf lives and impact

• They help cell membranes, or may used in some products

• They help stop gemiation in the body.

• Use to clean all the growing cells around

• They effect cell membrane function

• Control molds and bacteria in foods

• They effect some orgiansms of cell membrane, like Clotridum’s

• There can e carcinogens with high cell ammount

• Always limit intake